Project Summary Sepsis is a major health threat and the primary cause of morbidity and mortality in the critically ill patients with infection or trauma. Current treatment for sepsis is mainly supportive, including broad spectrum antibiotics, fluid resuscitation and vasopressor. Over the last 40 years, numerous attempts have been made to identify more specific and effective therapeutic strategies and pharmacological agents for treatment of sepsis. At present, there have been at least 150 clinical trials targeting various components in sepsis induction, including pathogen-associated molecular patterns (PAMPs), pattern-recognition receptors, and early cytokines or mediators produced in response to sepsis such as IL-1, TNF, C5a, and activated protein C. None has proven effective to date. Thus there is a critical need for targeted and effective sepsis therapy to reduce mortality from this disease. Most recent studies identified Gasdermin D (GSDMD) as a druggable target for the treatment of sepsis; however no specific inhibitors have been developed for this pathway. The objective of our research is to identify specific small molecule inhibitors of GSDMD via conducting a high throughput chemical genetic screening. The hypothesis is that these GSDMD inhibitors would alleviate both the early septic shock caused by systemic hyperinflammation and the following lethal secondary infections caused by immunosuppression, and thus should be potential pharmacological agents for preventing/treating sepsis. We have established a novel cell-based assay to measure and visualize GSDMD-induced lytic cell death. The preliminary data demonstrated the selectivity, reproducibility, and feasibility of our cell-based assay for high throughput screening. In this proposed research, we will first use this cell-based system to conduct a primary high throughput small molecule screening to identify a group of compounds that inhibit GSDMD-mediated lytic cell death (Aim I). Next, several secondary screening assays will be performed to confirm the effect of each positive hit compound identified from the primary screening and to identify the most specific and potent ones for future characterization (Aim II). In addition, we will directly examine the effect of positive compounds on macrophage and neutrophil function. The final lead compounds should be those that attenuate pyroptosis- mediated pro-inflammatory cytokine release from macrophages and delay neutrophil death, but do not affect GSDMD-independent functions such as phagocytosis and chemotaxis (Aim III). Finally, toward understanding the mechanism of action of each validated positive hit, we will examine whether the lead compounds can affect GSDMD-NT oligomerization and plasma membrane localization (Aim IV). GSDMD inhibitors identified in this study can be directly utilized as starting chemical compounds for novel sepsis drug development. Additionally, discovery of these inhibitors will greatly facilitate our research on the function and regulation of GSDMD in macrophages and neutrophils.